Adipogenic adenoviruses as a biomarker for disease

ABSTRACT

This invention relates to the relationship between infection with an adipogenic adenovirus, such as adenovirus-36, and obesity-related disease. In particular, this invention relates to assaying a subject to determine the adipogenic adenovirus infection status and then determining the subject&#39;s predisposition to developing an obesity-related disease based on the adipogenic adenovirus infection status.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.11/616,799, filed Dec. 27, 2006, which in turn is related to and claimsbenefit under 35 U.S.C. § 119(e) to Provisional Application Ser. No.60/753,402, filed Dec. 27, 2005, the disclosures of which are hereinincorporated in their entirety herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the relationship between infection withadipogenic adenoviruses, such as, for example, adenovirus-36 (Ad-36),and the etiologies of obesity and obesity-related cancers and otherdiseases. More specifically, the invention relates to a methodology fordetermining whether a subject is predisposed to developing anobesity-related disease due to an adipogenic adenovirus based on theadipogenic adenovirus infection status of the subject.

2. Related Art

There has been a dramatic simultaneous increase in the prevalence ofobesity and of certain types of cancer. A worldwide epidemic of obesityaccelerated dramatically starting about 1980. In the USA the prevalenceof obesity in adults more than doubled in the 20 years from 1980 to 2000(from 15% to 31%), whereas the prevalence increased only slightly in theprior 20 years from 1960 to 1980 (from 13.5% to 15%). The prevalence ofobesity in children tripled from about 1970 to 2000. Likewise, cancersof the breast, prostate, colon, and liver have also rapidly increased inprevalence in recent years.

Changes in reproductive hormones in obesity have been suggested to playa role in the association of breast and prostate cancer (among others)and in the aggressiveness of these cancers. However, changes inreproductive hormones cannot explain cancers such as colon, renal, orpancreatic cancer that are not under hormonal control.

Another possibility for the link between obesity and cancer is thedecreased immune function seen in obese individuals. Obese people have alower antibody response to vaccination with hepatitis B than vaccinepeople who are not obese. The immune system is critical in inhibitingthe growth of neoplasms, so it would not be surprising if this were themechanism of increased cancers of many types in obesity. However,adenoviruses are well known to decrease immune function as a way toenhance their replication within the host, including human hosts. Morerelevant, the SMAM-1 avian adenovirus, which has been reported to causeobesity, had a major impact by decreasing immune function of chickens.Thus, there is a direct link between an adenovirus that causes obesityand that also impairs immune function. The inventor has reported thatSMAM-1 is associated with obesity in humans, adding a link fromadenovirus to human obesity.

Some human adenovirus serotypes are known to be oncogenic, and inducetumors in rats or hamsters. Adenovirus serotypes are divided by Groups.Group A adenoviruses (e.g., Ad-12, Ad-18) are highly oncogenic,producing tumors in most animals within 4 months; group B adenoviruses(e.g., Ad-3, Ad-7) are weakly oncogenic, inducing tumors in most animalswithin 4 to 18 months; group D viruses are thought to be less oncogenic,but serotype-9 efficiently induces mammary tumors within 3 to 5 months.There has been extensive work on adenovirus-induced cancer, but to date,there has been no evidence that adenoviruses cause human cancer.

If adenoviruses cause human cancers, it is likely that they do so byaltering expression of genes in the host that allow unregulated cellgrowth to occur. Many such tumor markers have been identified. Some aredue to genetic variants. Hereditary breast cancer has been linked togermline mutations in one allele of high penetrance susceptibility genessuch as BRCA1, BRCA2, CHEK 2, TP53 or PTEN. It is possible thatadenovirus infections facilitate cancer in these genetically susceptibleindividuals. However, adenoviruses may contribute to spontaneousoncogenesis by inducing expression of various oncogenes or suppressingexpression of tumor suppressor genes of the host. Among the alterationsdue to adenoviruses that are thought to contribute to cancer are changesin DNA-dependent protein kinase, fatty acid binding protein, mTOR, p16,p53, PDZ protein, phosphatidylinositol 3-kinase, PML, thymidine kinase,and Zip kinase. Of particular interest are those tumor related factorsthat are altered by the adenovirus E4 region, and specifically theE4orfl gene. The E4 oncogenes include DNA-dependent protein kinase, p53,PDZ protein, phosphatidylinositol 3-kinase, PML, thymidine kinase, andZip kinase. As will be described below, the Ad-36 E4orfl gene has beenshown to be involved in producing obesity by a direct effect onadipocyte metabolism. The E4orfl region of human adenovirus-5 has beenshown to be an oncogene, and a recent paper reported that Ad-5 producesobesity in mice. These findings show a direct link between obesity andcancer, with both being due to a human adenovirus, and provide thelikely mechanism via a viral gene.

The general consensus of many investigators in the field of cancerresearch has been that adenoviruses do not contribute to the etiology ofhuman cancer. Therefore, it would be a major technological advance todemonstrate that adipogenic adenoviruses, such as Ad-36, are associatedwith obesity-related cancers, such as breast and prostate cancers.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for determining if a subject ispredisposed to developing an obesity-related disease due to anadipogenic adenovirus. The method includes obtaining a sample from thesubject, assaying the sample to determine whether the subject isinfected with the adipogenic adenovirus and determining that a subjectinfected with an adipogenic adenovirus is predisposed to developing anobesity related disease due to an adipogenic adenovirus relative to asubject not infected with an adipogenic adenovirus. The subject may behuman or animal. The invention may be implemented in a number of ways.

[TTC to Complete when Claims are Finalized]

Additional features, advantages, and embodiments of the invention may beset forth or apparent from consideration of the following detaileddescription, drawings, and claims. Moreover, it is to be understood thatboth the foregoing summary of the invention and the following detaileddescription are exemplary and intended to provide further explanationwithout limiting the scope of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a furtherunderstanding of the invention, are incorporated in and constitute apart of this specification, illustrate embodiments of the invention andtogether with the detailed description serve to explain the principlesof the invention. No attempt is made to show structural details of theinvention in more detail than may be necessary for a fundamentalunderstanding of the invention and various ways in which it may bepracticed.

FIG. 1 is a BODIPY staining of 3T3-L1 cells 5 days post MDI treatment.This figure shows triglycerides in 3T3-L1 cells infected with Ad-36versus uninfected in vitro. Control cells show a moderate BODIPY fatstain whereas Ad-36 infected cells have about twice as muchtriglyceride, showing adipocyte biochemistry has changed.

FIG. 2 is a graph showing the effects of spontaneous infection withAd-36 in ad libitum fed rhesus monkeys. As compared to the period beforea monkey became infected, once infection was noted (designated T) therewas a steady rise in body weight that was still rising at about 18months.

FIG. 3 is a graph showing an increase in body weight in monkeys infectedwith Ad-36. As compared to uninfected monkeys, infected monkeys gainedabout four times as much weight in about seven months.

FIG. 4 is a graph showing a decrease in serum cholesterol in Ad-36infected marmoset monkeys. Serum cholesterol began dropping immediatelyafter infection and by about 10 weeks. This is significantly differentthan baseline in infected monkeys. There is no change in uninfectedmonkeys.

FIG. 5 is a gel showing the presence of Ad-36 DNA in adipose tissue ofinfected marmosets using a nested PCR assay. Lane 1 is Ad-36 DNA, lanes2-4 show no Ad-36 DNA in fat of uninfected monkeys, and lanes 5-7 showsthe presence of Ad-36 DNA in all infected monkeys.

FIG. 6 is gel showing Ad-36 DNA in liver (upper lanes) and muscle tissue(lower lanes) of infected marmosets using nested PCR assay. Ad-36 DNAfrom infected monkeys is seen in lanes 7-9 of liver tissue and muscletissue and is not present in lanes 4-6 from uninfected monkeys. Lane 2is Ad-36 from culture, lane 3 is marker.

FIG. 7 is a gel showing Ad-36 DNA in brain and muscle tissue of infectedanimals by nested PCR assay. Upper lanes are brain tissue frommarmosets, lower lanes are muscle tissue. Ad-36 DNA from infectedmarmosets is seen clearly in lanes 8-9 and more faintly in lane 7. Ad-36DNA is not present in lanes 4-6 from non-infected marmosets. Lane 2 ispositive control from Ad-36 culture.

FIG. 8 is a gel showing Ad-36 DNA in adipose tissue of humans usingnested PCR assay (bottom lanes). This gel shows Ad-36 DNA in adiposetissue of marmosets in the upper lanes (repeat of data in FIG. 5: lanes4-6 negative from control marmosets, lanes 7-9 Ad-36 DNA from infectedmarmosets). In bottom lanes Ad-36 DNA is seen in 2 of 6 samples of humanvisceral adipose tissue obtained from cadavers at autopsy (lane 2 ispositive control, lanes 6-7 are Ad-36 positive, lanes 4, 5, 8, 9 arenegative).

FIG. 9 is a chart showing the effects of multiple human adenoviruses ontotal body fat in chickens. Ad-2 and Ad-31 did not increase body fat,but Ad-37 had a marked effect.

FIG. 10 is a chart showing the effects of multiple human adenoviruses onvisceral fat in chickens. Ad-2 and Ad-31 did not increase visceral fat,but Ad-37 had a marked effect.

FIG. 11 is a chart showing food intake in animals exposed to multiplehuman adenoviruses. There were no differences in cumulative food intakeamong groups, yet individuals infected with Ad-37 became obese whileindividuals infected with Ad-2 or Ad-31 were not.

FIG. 12 is a table showing human Ad-5 produced obesity in mice. Also,body fat increased by almost 3 fold.

FIG. 13 is a table showing the presence of serum antibodies to humanAd-36 in people from three US cities. An average of about 30% of obesepeople were infected with As-36 versus about 11% of non-obese who werenot infected with Ad-36.

FIG. 14 is a chart showing body mass index in 502 individuals from threeUS cities according to status of infection with Ad-36. Overall, BMI wasabout 9 units higher in infected versus uninfected people (p<0.0001).Infected individuals of both obese and non-obese groups weresignificantly heavier than the uninfected in each group.

FIG. 15 is a table showing a comparison of twin pairs discordant forinfection with Ad-36. Out of 89 twin pairs, 26 were discordant. Thetwins infected with Ad-36 were heavier and fatter than their uninfectedco-twins.

FIG. 16 is a gel showing the presence of Ad-36 DNA in prostate cancertissue.

FIG. 17 is a gel showing the presence of Ad-36 DNA in monkeys.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methodology, protocols, devices, apparatus, materials, andreagents, etc., described herein, as these may vary. It is also to beunderstood that the terminology used herein is used for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention. It must be noted that as used herein andin the appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “a virus particle” is a reference to one ormore virus particles and equivalents thereof known to those skilled inthe art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Preferred methods, devices,and materials are described, although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention. All references cited herein areincorporated by reference herein in their entirety.

Any numerical values recited herein include all values from the lowervalue to the upper value in increments of one unit provided that thereis a separation of at least two units between any lower value and anyhigher value. As an example, if it is stated that the concentration of acomponent or value of a process variable such as, for example, dosage,dilution, and the like, is, for example, from 1 to 90, specifically from20 to 80, more specifically from 30 to 70, it is intended that valuessuch as 15 to 85, 22 to 68, 43 to 51, 30 to 32 etc., are expresslyenumerated in this specification. For values which are less than one,one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate.These are only examples of what is specifically intended and allpossible combinations of numerical values between the lowest value andthe highest value enumerated are to be considered to be expressly statedin this application in a similar manner.

Moreover, provided immediately below is a “Definition” section, wherecertain terms related to the invention are defined specifically forclarity, but all of the definitions are consistent with how a skilledartisan would understand these terms. Particular methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention.

DEFINITIONS

Ad-2 is adenovirus type 2

Ad-5 is adenovirus type 5

Ad-31 is adenovirus type 31

Ad-36 is adenovirus type 36

Ad-37 is adenovirus type 37

BMI is body mass index

FAS is fatty acid synthetase

PPAR is peroxisome proliferator activated receptors

CEBP is CCAAT-enhancer binding protein

The term “adipogenic adenovirus” as used herein generally refers toadenoviruses that are capable of stimulating increase lipid productionin cells, tissues, and/or organs by turning on the cellular machinery ininfected hosts to turn on the host's production of lipogenic enzymeswhich then produce excess fatty acids and promote fat storage with theinfected cells. The adipogenic adenoviruses include without limitationAd-5, Ad-36, and Ad-37.

The term “BMI” as used herein generally refers to a statistical measureof the weight of a person scaled according to height. BMI may be definedas the individual's body weight divided by the square of the height andmay be expressed in the unit kg/m². BMI may be used as a screening toolto identify possible weight problems for adults and children. However,in order to determine if excess weight is a health risk, a healthcareprovider may need to perform further assessments, such as skinfoldthickness measurements, evaluations of dies, physical activity, familyhistory, hip to waist ratio, infection with a adipogenic adenovirus, andother appropriate health screenings. For adults 20 years old and older,BMI may be interpreted using standard weight status categories that arethe same for all ages and for both men and women. Alternatively, forchildren and teens, the interpretation of BMI is both age- andsex-specific. For example, an adult having a (i) a BMI less than about18.5 percent mat be considered underweight, (ii) a BMI is the range ofabout 18.5 to about 24.9 may be considered normal weight, (iii) a BMI inthe range of about 25 to about 29.9 may be considered overweight, and(iv) a BMI greater than about 30.0 may be considered obese.

The term “hip to waist ratio” refers to a measurement that may be usedto help determine obesity. The distribution of fat is evaluated bydividing the waist size by the hip size. For example, an individual withabout a 30 inch waist and about a 40 inch hip size would have a ratio ofabout 0.75 and an individual with about a 41 inch waist size and about a39 inch hip size would have a ratio of about 1.05. The higher the ratio,the higher the risk of heart disease and other obesity-relateddisorders.

A “biological sample” refers to a sample of tissue or fluid from a humanor animal including, but not limited to plasma, serum, spinal fluid,lymph fluid, the external sections of the skin, respiratory, intestinaland genitourinary tracts, tears, saliva, blood cells, tumors, organs,tissue and sample of in vitro cell culture constituents.

An “isolated” or “substantially pure,” nucleic acid (e.g., DNA, RNA, ora mixed polymer) for example, is one which is substantially separatedfrom other cellular components which naturally accompany a native humanor animal sequence or protein, e.g., ribosomes, polymerases, many otherhuman or animal genome sequences and proteins. The term embraces anucleic acid sequence or protein which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogs or analogs biologicallysynthesized by heterologous systems.

The term “immunogenic,” generally refers to an anti-obesity vaccine thathas the capability to provoke in an immunized animal, an immune responsethat yields neutralizing antibodies against an obesity-causing, livevirus that might infect the person after administration of the vaccine.

The term “antibody” refers to antibodies, digestion fragments, specifiedportions and variants thereof, including antibody mimetics or comprisingportions of antibodies that mimic the structure and/or function of anantibody or specified fragment or portion thereof, including singlechain antibodies and fragments thereof. The invention encompassesantibodies and antibody fragments capable of binding to a biologicalmolecule (such as an antigen or receptor), such as the fiber coatprotein of adenoviruses, and specifically, Ad-36, or portions thereof.

The term “nucleic acid sequence,” includes an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material, natural or synthetic inorigin.

Fragments: include any portion of a heterologous peptide or nucleic acidsequence. Heterologous peptide fragments retain at least one structuralor functional characteristic of the subject heterologous polypeptides.Nucleic acid sequence fragments are greater than about 60 nucleotides inlength, and most preferably includes fragments that are at least about100 nucleotides, at least about 1000 nucleotides, and at least about10,000 nucleotides in length.

Complementary or complementarity: as used herein, include the naturalbinding of polynucleotides under permissive salt and temperatureconditions by base-pairing. For example, the sequence “A-G-T” binds tothe complementary sequence “T-C-A.”

Complementarity between two single-stranded molecules may be “partial,”in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands andin the design and use of molecules.

Functional equivalent: a protein or nucleic acid molecule that possessesfunctional or structural characteristics that are substantially similarto a heterologous protein, polypeptide, enzyme, or nucleic acid. Afunctional equivalent of a protein may contain modifications dependingon the necessity of such modifications for the performance of a specificfunction. The term “functional equivalent” is intended to include the“fragments,” “mutants,” “hybrids,” “variants,” “analogs,” or “chemicalderivatives” of a molecule.

Protein purification: broadly defined, any process by which proteins areseparated from other elements or compounds on the basis of charge,molecular size, or binding affinity.

Substantially purified: as used herein, includes nucleic or amino acidsequences that are removed from their natural environment, isolated orseparated, and are at least 60% free, preferably at least 75% free, andmost preferably at least 90% free from other components with which theyare naturally associated.

Inhibition: as used herein, refers to a reduction in the parameter beingmeasured, whether it be adenovirus type 36 growth or viability. Theamount of such reduction is measured relative to a standard (control).“Reduction” is defined herein as a decrease of at least around 25%relative to control, preferably at least around 50%, and most preferablyof at least around 75%.

The anti-adipogenic adenovirus vaccines of the invention, wherein theimmunogenic component is live, inactivated virus, killed virus, coatprotein per se, epitope-comprising coat protein segment, or coat protein(or epitope-comprising segment thereof) provided with use of anon-pathogenic, genetically modified carrier virus such as a vacciniavirus or a fowl pox virus, are prepared using methods well known in theart. Thus, the vaccines will include carriers, excipients, adjuvants,antimicrobials, preservatives and the like as well understood in theart. Thus, in addition to the active ingredient, the vaccines will havesuitable compositions, usually aqueous buffers, such asphosphate-buffered saline or the like, in which the active ingredientwill be suspended along with, optionally, any of various immune-systemstimulating adjuvants used in human vaccine preparations, antimicrobialcompositions, and other compositions to stabilize the preparations. Allcompositions included with the vaccine preparation will be suitable foradministration to humans. The vaccine preparation may be stored inlyophilized form and then combined with solution soon beforeadministration. For oral administration, the vaccine preparation may bein solution, tablet or pill form optionally with an enteric coating asunderstood in the art. The concentration of active (immunogenic orimmunogen-providing) component in solution with which it is administeredtypically will be between about 1 ng and about 1 mg/ml.

The anti-adipogenic adenovirus vaccines of the invention will beadministered intranasally, orally, or by injection intravenously,intramuscularly, subcutaneously or peritoneally. Administration of thevaccines of the invention is to be under the guidance of a physician.

Appropriate dosing of the anti-adipogenic adenovirus vaccine is wellwithin the skill of medical practitioners and will depend on a number offactors including the age of the person being treated, the urgency ofthe person's developing protective immunity, the status of the person'simmune system, and other factors known to the skilled. The vaccinetypically will be administered in several steps in order to cause andmaintain protective immunity against obesity-causing virus in the personbeing vaccinated. Thus, after the primary vaccination, there typicallywill be between one and about ten booster vaccinations separated byperiods between about 1 week and 10 years.

A single dose of an anti-obesity vaccine of the invention (in solutionform) will have a volume of between about 0.1 ml and 10 ml and, in anyform, will have between about 1 ng and 10 mg of killed or inactivatedadipogenic virus, between about 1 ng and 10 mg of genetically modified,non-pathogenic virus, or between about 1 ng and 10 mg of coat protein(e.g., fiber protein) or 6-30 amino acid peptide (in its form asmodified to be immunogenic).

An anti-adipogenic adenovirus vaccine of the invention, wherein theactive ingredient is nucleic acid, will also be a standard preparationfor vaccines of that type. With vaccines of this type, the nucleic acidis not the immunogen but is expressed in vivo after administration ofthe vaccine as a peptide or protein which in turn is immunogenic.Vaccines of this type will be administered by techniques known in theart for such vaccines (e.g., intramuscular injection). Dosing will alsobe according to procedures known in the art to cause and maintainprotective immunity against viral obesity in the vaccinated individual.

Note that an anti-adipogenic adenovirus vaccine according to theinvention may include active ingredients based on more than oneadipogenic virus (or the coat protein (e.g. fiber protein) or epitopicsegments of the coat protein thereof).

In yet another aspect, the invention is a method of preventingadipogenic adenovirus related disease caused by a virus in a humansusceptible thereto which comprises administering to the human an amountof an anti-adipogenic adenovirus vaccine of the invention that iseffective to raise and maintain a protective immune response against anadipogenic adenovirus.

This invention generally relates to the relationship between infectionwith human adipogenic adenoviruses, such as Ad-5, Ad-36, and Ad-37, theetiologies of obesity, and the dysfunction of cells, tissues and/ororgans, such as cancers, dysfunction of the pancreas, dysfunction ofskeletal and cardiovascular muscle, pulmonary dysfunction, dysfunctionof the brain and nervous system, and adrenal disease and dysfunction.Moreover, the invention relates to utilizing adipogenic adenovirusstatus as a predictor for disease status, prognosis, treatment outcome,and prevention of infection and disease.

The main mechanism of adipogenic adenovirus-related disease is theproduction of lipids within cells. The adipogenic adenoviruses arecapable of changing the cellular machinery in infected subjects to turnon the host's production of lipogenic enzymes. As a result, thelipogenic enzymes make excess fatty acids and promote fat storage withinthe cells of multiple organs.

Multiple cells of the body are known to have the capacity to make fattyacids within their cells. Fatty acid synthetase (FAS) is one of the mostimportant lipogenic enzymes and is expressed in a number of adult andfetal cells, which suggests that these tissues are capable of producingfatty acids. For example, FAS is expressed in adult cells such asepithelial cells of the duodenum and stomach, hemopoietic cells,appendix, ganglion cells of alimentary tract, hepatocytes, mast cells,seminal vesicle, umbrella cells of urinary bladder, adrenal zonafasciculate cells, adipocytes, anterior pituitary cells, basket cells ofcerebellum, cerebral cortical neurons, deciduas, decidualized stromalcells of endometrium, epithelial cells of apocrine gland, duct andacinus of breast, prostate, and sebaceous gland, letein cells, and TypeII alveolar cells of lung. Additionally, FAS is also expressed in fetalcells such as anterior pituitary cells, chondrocytes of tracheobronchialwall, endothelium of blood vessels and heart, epithelial cells ofbronchus, esphagogastrointestinal tract, lung, pancreas, prostate,thyroid, tongue, trachea, proximal tubules of kidney, fibroblasts, nodallymphocytes, neuroblasts in adrenal medulla, thymocytes, striatedmyocytes of tongue, epithelial cells of salivary glans andtracheobronchial glands, hemopoietic cells, heptocytes, Lanhans cells ofchorionic villi, osteoblasts, perivertebral fibroblastic cells, Schwanncells of sympathetic ganglion and Auerbach plexus, subcapsular cells ofadrenal, adipocytes, Leidig cells of testis, mast cells, uroepitheliumof urinary tract, and adrenocortical cells of upper layer.

While fatty acids are critical to the intracellular milieu for manybiochemical processes, excess fatty acids within the cells profoundlyalter cellular biochemistry. Excess fatty acids within cells may lead toabnormal functioning of a number of intracellular processes. Forexample, excess fatty acids in pancreatic tissue reduce insulinsecretion, and fatty acids in cancer cells provide the major source ofenergy for cancer growth. Moreover, fatty acids within the lung cellsstimulate attraction of macrophages to the area and are associated withasthma and emphysema. Therefore, adverse results occur when cells areexposed to excess to fatty acids.

According to one embodiment adipogenic adenoviruses may be a mechanismby which excess fatty acids and triglycerides are produced in cells andtissues. Studies have demonstrated that adipogenic adenoviruses canalter lipogenic enzymes in adipose tissue and in liver. For example,other studies have shown that infection with the adipogenic adenovirus,Ad-36, stimulates the rapid appearance of differentiation factorsincluding glycerol-3-phosphodehydrogenase, PPAR-gamma, CEBP-alpha andbeta, and lipoprotein lipase in 3T3-L1 cells. Since multiple tissuesaccumulate adipogenic adenoviral DNA, excess lipogenic enzymes may bepresent in all infected tissues that are capable of producing theseenzymes.

According to another embodiment of the invention, adipogenic adenovirusinfection may be used to determine the presence of an obesity-relatedcancer. In a further embodiment, adipogenic adenovirus infection may beused to determine whether a subject is predisposed to developing anobesity-related cancer.

The prevalence of multiple types of cancers is increased in obesepeople. These cancers include without limitation breast, prostate,uterus, ovary, colon, kidney, pancreas and lung. Hepatocellular cancermay also be linked to obesity and the metabolic syndrome. Additionally,obesity may be linked to the aggressiveness of some types of cancer andto a poorer prognosis. For example, obesity may be associated with ahigher grade of prostate cancer and higher recurrence rates afterradical prostatectomy. Also, in non-Hispanic White women, breast tumorsize correlates with obesity. Specifically, this is most notable for thehighest quartile of waist circumference where the odds ration is 2.76.

Studies have shown that fatty acids are the substrate for energyexpenditure in cancer cells and that blocking FAS can block cancergrowth. Moreover, it has been shown that FAS is increased in manyobesity-related cancers and that FAS favors tumor growth. Adipogenicadenovirus infection in cancer cells may stimulate fatty acids in cellthereby promoting cancer growth by providing an energy source for thecancer cells.

FAS expression has been detected in various tumors and associated withhistological subtype, histopathological grade and tumor aggressiveness.Studies have demonstrated that FAS is highly expressed in human neoplasmsuch as breast, prostate, ovarian, colorectal, and endometrial cancers.It has been determined that FAS (OA-519) was a predictor of prostatecancer. It was observed that OA-519 immunoreactivity was seen in 56(57%) of the 99 primary prostate cancers examined. OA-519 cancers weremore likely to progress than the non-OA-519 cancers.

Indeed, the presence of Ad-36 was confirmed in 61% of human prostatecancer tissues and it was observed that 50% and 51% of breast andprostate cancer patients, respectively, have antibodies to Ad-36 incomparison to 13.8% of the normal population.

Therefore, the presence of FAS in prostate cancer tissue inapproximately the same number of cancer patients as were observed to beinfected with Ad-36 in combination with the findings that adipogenicadenoviruses increase FAS in cells and that inhibition of FAS mayinhibit cancer, suggest that infection of an adipogenic adenovirus causeboth obesity and obesity-related cancer in a subject.

According to an embodiment of the invention, the prevalence of Ad-36infection may be statistically higher in patients with breast andprostate cancer than in individuals without cancer. In a furtherembodiment, Ad-36 status may serve as a marker for breast and prostatecancer. Yet a further embodiment relates to patients with breast orprostate cancer who have Ad-36 DNA in their tissues will have moreaggressive cancer and/or a poorer prognosis than those without Ad-36DNA.

According to another embodiment of the invention, adipogenic adenovirusinfection may be used as a predictor for disease prognosis, treatmentprognosis, or as a predictor of tumor aggressiveness. In breast,prostate and ovarian carcinomas, high levels of FAS are often associatedwith poor prognosis. The association between FAS expression and tumorsize or a marker of proliferative activity of tumor cells may suggestthat FAS is related to growth and proliferation of these malignanttumors.

For example, studies have shown that overexpression of FAS increasedheptacarcinogenesis in rat. Other studies have shown that about 30% ofpatients with early breast cancers expressed FAS. The patients withbreast cancer expressing FAS had significantly higher tumor grade,larger tumor volume, advancing clinical stage, and Gleason's score, oneof the most powerful predictors. FAS expression has also been studied inovarian neoplasia, where it was seen to be associated with histologicaltumor grade and shorter survival. Moreover, studies have shown thatoverweight and obese individuals had significantly increased mortalityfrom multiple types of cancer in humans.

FAS is associated with colon cancer and with its severity. Studies haveshown that only 2 (5%) of 43 adenomas with low-grade dysplasia showedreactivity for FAS. However, positive FAS immunostaining was seen in 7(17%) of 40 cases of adenomas with moderate-grade dysplasia, in 9 (53%)of 17 cases of adenomas with high-grade dysplasia, and in 81% ofadenocarcinomas (p<0.0001).

Therefore, one embodiment of the invention is directed to a diagnosticscreening test for the presence of adipogenic adenovirus infection in asubject. If the subject tests positive for adipogenic adenovirus, FASexpression may be increased in cancer cells resulting in a moreaggressive cancer and/or a poorer prognosis. Other information linkingAd-36 and cancer is the finding of unique sequences of Ad-36 DNA inobesity-related cancers, such as prostate cancer tissue by polymerasechain reaction test (PCR). The unique sequences of DNA from Ad-36 fiberprotein, which are described in Assignee's previous U.S. Pat. Nos.6,664,050 and 6,127,113 were detected in 11 of 18 samples of prostatecancer tissue. Exemplary screening techniques are described below.

The adipogenic adenovirus test will be useful in two ways. If a subjectwith a breast lump or an enlarged prostate has a positive adipogenicadenovirus test (tested by, for example, either a positive serum test ora positive PCR tissue test), there is a higher risk that the subject hascancer and tests should be done to evaluate for cancer (e.g., biopsy)rather than “watchful waiting.” Conversely, if a person has a positiveadipogenic adenovirus test, then the subject should be screened forcancer at more regular intervals than a person with a negative Ad-36test.

A vaccine against Ad-36 has been developed in rabbits using killedvirus. Serum from the rabbits contains antibodies that prevent Ad-36growth in tissue culture in up to 17 serial dilutions (a dilution of1:131,072 of the original concentration). It is known that the onlyeffective antibodies against adenovirus growth are directed againstsegments of fiber protein of the various adenoviruses (neutralizingantibodies). Ad-36 antibodies do not cross react with any otheradenoviruses, so unique DNA sequences in Ad-36 fiber protein areresponsible for this specificity. Three sequences in Ad-36 fiber proteinthat are unique to Ad-36 have been identified. Using standard technologyin the field, peptides from these sequences will be used to make ahighly purified vaccine that should have minimal allergic reactions. Thepeptides will be bound to adjuvants commonly used in vaccines to enhanceeffectiveness. These techniques are known by those of skill in the art.

In a particular embodiment, a vaccine against adipogenic adenoviruses,such as Ad-36 to prevent cancer may be made. Since adipogenicadenoviruses are associated with cancer, a vaccine against adipogenicadenoviruses may be used to prevent cancer. For example, a vaccineagainst Ad-36 using unique DNA sequences contained in the fiber proteinDNA to make peptides may be developed. This technology is known by thoseof skill in the art.

In a further embodiment, a novel concept of sequencing the fiber proteinDNA from all of the adipogenic adenoviruses to determine theirstructures may be performed. Following sequencing, the DNA sequencesthat are present in many or all of the fiber proteins will be identifiedusing DNA comparison databases commonly used in the field. This may beaccomplished by, for example, employing GenBank to identify the uniquesequences in Ad-36 fiber protein DNA. DNA sequences of about 30-75 basepairs that code for peptides of about 10-25 amino acids are thenselected. It is known that peptides in the knob portion of the fiberprotein are effective, so these sequences will be examined. Followingidentification, the peptides may be coupled with adjuvants to make avaccine that will prevent infection from any of the human adenoviruses.The usefulness of a common vaccine for the adipogenic adenoviruses willbe to prevent obesity, cancer, and other human diseases that are due toadipogenic adenoviruses. For example, a significant percentage ofmilitary recruits contract adenovirus infections during basic training.Also, many of the mild illnesses associated with fever, cough, diarrhea,or conjunctivitis in the first two years of life are due toadenoviruses.

According to one embodiment of the invention, adipogenic adenovirusinfection may be used as a predictor of the development of obesity anddiseases due to the complications of obesity and the outcome of regimensthat affect body weight. Complications of obesity may include, interalia, diabetes mellitus, hypertension, hyperlipoproteinemia, cardiacdisease such as atherosclerotic disease and congestive heart failure,pulmonary diseases such as sleep apnea and asthma, cerebrovascularaccidents, cancers such as breast, uterus colon and prostate cancer,gall bladder disease such as stones and infection, toxemia duringpregnancy, risks during surgery, gout, decreased fertility, degenerativearthritis, and early mortality.

It has been demonstrated by the Assignee that human Ad-36 stimulates thestorage of fat and formation of new fat cells in mouse preadipocytes(3T3-L1 cells) (FIG. 1) and in human preadipocytes. Additionally,Assignee has demonstrated in U.S. Pat. Nos. 6,127,113 and 6,664,050,herein incorporated in their entirety by reference, that Ad-36 infectionis associated with obesity. For example, four experiments were conductedin chickens, one experiment in mice, and two experiments in monkeys, allshowing that infection with Ad-36 increased body fat and lowered serumcholesterol and triglycerides. Most notable, food intake measured inchickens, mice, and rats was not difference between infected and controlanimals indicating that energy expenditure was different. The mechanismof Ad-36 has a direct effect on adipocytes to increase lipogenic enzymesand differentiation factors. Cells infected with Ad-36 exhibited about 2times as much stored triglyceride at 5 days.

A large number of studies were also performed with the adipogenicadenovirus, Ad-36. Rhesus monkeys that were spontaneously infected withAd-36 showed a significant weight gain in 18 months after infection(FIG. 2). Marmosets were also infected with Ad-36 and a four-fold weightgain was observed in infected marmosets in comparison to uninfectedmarmosets (FIG. 3). Moreover, body fat increased by about 70% and serumcholesterol dropped by about 35 mg/dl after infection (FIG. 4). Notably,Ad-36 DNA was observed in multiple tissues of the monkey, such as brain,liver, lung, muscle and adipocyte tissue seven months following initialinfection and was no longer able to grow the virus from the blood orfeces of the infected monkeys after two months (FIGS. 5-7). Ad-36 DNAhas also been observed in human adipocyte tissue as seen in FIG. 8.

Other studies have demonstrated that Ad-37 caused obesity in chickens,but Ad-2 and Ad-31 did not (FIGS. 9-11). Cumulative food intake was notdifferent among the groups studied. Moreover, studies have alsodemonstrated that Ad-5 caused obesity in mice (FIG. 12). These studiesshow that stimulation of lipogenic enzymes and obesity are notnon-specific effects of all adenovirus infections, but multipleadenoviruses may do so.

Another embodiment of the invention includes using adipogenicadenoviruses to predict diabetes and other pancreatic dysfunction.Additionally, another embodiment is directed to a vaccine againstadenoviruses that may prevent pancreatic disease, including some casesof diabetes.

Pancreatic beta cells make lipids and lipid metabolism in the beta-cellis critical for the regulation of insulin secretion. Enzymes that areassociated with lipid synthesis or oxidation cause a decrease in theamount of insulin produced and secreted. Studies have demonstrated thatoverexpression of SREBP-1 in beta cells decreases insulin synthesis.SREBP-1 is a direct precursor of FAS and has been found to be increasedby adenovirus infection in vitro and in vivo in hepatocytes. Otherstudies have demonstrated that carbohydrate responsive element-bindingprotein (ChREBP) binding to fatty acid synthase and L-type pyruvatekinase genes is stimulated by glucose in pancreatic beta-cells. Highercarnitine palmitoyltransferase I (CPT I) protein levels in beta cellscauses a decrease in secretion of insulin in response to glucose.Adipogenic adenoviruses may cause diabetes because they increase obesityand produce insulin resistance, but the changes in lipogenic enzymes dueto virus infection of the pancreas also may play a role.

An additional embodiment of the invention is for using adipogenicadenoviruses status to predict liver disease, cirrhosis, and other liverdysfunction. Also, a vaccine against adenoviruses may be used to preventliver diseases due to adipogenic adenoviruses.

Obesity is associated with liver disease. The spectrum of liver diseasewith obesity starts with fat infiltration of the liver, progresses tosteatohepatitis (non-alcoholic steatohepatitis=NASH), and a sizeablepercentage of patients with NASH go on to develop cryptogenic cirrhosis.The frequency and severity of fatty infiltration of the liver goes upwith increasing body weight and is present in the vast majority ofpatients with morbid obesity. It has been reported that obesity ispresent in about 30% to about 100% of the cases of non-alcoholic fattyliver disease (NAFLD). Other studies have demonstrated that greater thanabout 95% of subjects with severe obesity had various degrees of fattyinfiltration of the liver and about 65% had NASH.

Adipogenic adenoviruses produce increased levels of lipogenic enzymesand an accumulation of fat in the liver. As noted above, fattyinfiltration of the liver may lead to cirrhosis. There are a number ofcauses of fatty infiltration of the liver, and additional insults to theliver may increase the chance that cirrhosis will occur. Individualswith risk factors for liver disease or with abnormal liver functiontests should be tested for adipogenic adenoviruses as they may needspecial attention to prevent progression of fatty liver to cirrhosis. Avaccine against adipogenic adenoviruses may reduce or prevent many casesof fatty liver and cirrhosis.

Yet another embodiment of the invention includes using adipogenicadenoviruses to predict muscle dysfunction. Also, a vaccine againstadenoviruses may be used to prevent muscle dysfunction.

Muscles are known to have lipogenic enzymes present and to storeintracellular lipids. Obesity is associated with abnormal lipidmetabolism and accumulation of intramuscular lipid. Studies have shownthat a lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is up-regulatedin skeletal muscle from extremely obese humans. High intramuscular fatis related to poor muscle function in obese people and is related toinsulin resistance and diabetes. Other studies have showed that insulinresistance and type 2 diabetes result from the accumulation of lipids intissues not suited for fat storage, such as skeletal muscle and theliver. FFAs stimulated the de novo synthesis of ceramide andsphingosine, two sphingolipids shown previously to inhibit insulinaction. The inability to transition from fat to glucose as the primarysource of fuel may be associated with insulin resistance, metabolicdysregulation, and cardiovascular risk. Since leptin has been shown togo down in adipocytes infected with Ad-36, this might produce insulinresistance.

Obese people complain of pain with exercise. The reasons for this arenot clear, but obese individuals have more intramuscular lipids and alower exercise capacity. The intramuscular lipids alter the substrateutilization of skeletal muscles. Increased intramuscular lipids due toadipogenic adenoviruses may limit exercise capacity, increase pain, andalter substrate utilization. This would be particularly important inanimals used for performance or endurance, such as race horses or racingdogs. An animal (or person) infected with an adipogenic adenovirus maynot be able to achieve as good a performance as an uninfected, sotesting for adipogenic adenoviruses may be used as a predictor ofperformance quality.

Finally, increased intramuscular lipid in heart muscle may alter cardiacfunction, produce cardiomyopathy and congestive heart failure. Studieshave showed that accumulation of excess fatty acids and triglycerideswithin heart muscle is associated with cardiac insulin resistance andcardiac dysfunction.

A further embodiment of the invention is related to using adipogenicadenoviruses status to predict pulmonary dysfunction. Also, a vaccineagainst adenoviruses will prevent pulmonary dysfunction.

It has been known for many years that obesity is associated withpulmonary disease such as asthma and emphysema. The mechanisms of thisassociation are not clear. It is known that the lungs make FAS andsynthesize lipids. Therefore, the finding that adipogenic adenovirusescan cause lung disease may be associated with the production of FAS inthe lungs by adenoviruses. Studies have indicated that Ad-5 infectiondoubles the number of macrophages in the lung of guinea pigs. Thecombination of smoking and Ad-5 quadruples the number of macrophages.Latent viral infections were present in patients with asthma andemphysema and Ad-5 ElA protein in the lungs was correlated withinflammation in asthma and emphysema patients.

An embodiment of the invention is using adipogenic adenovirus status topredict brain and nervous system dysfunction. Also, a vaccine againstadenoviruses may be used to prevent brain and nervous systemdysfunction.

Adipogenic viruses infect brain tissue. It is well known that the nervesand brain make FAS. Obesity is linked to adverse neurocognitive outcome,including reduced cognitive functioning and Alzheimer's disease. Studieshave demonstrated that BMI was inversely related to performance on allcognitive tests and there was no evidence of a BMI×age interaction.There is an increased prevalence of Alzheimer's disease in obesity.Abeta42, a type of amyloid that is deposited in Alzheimer's patients,correlated with BMI. Obese people may have about a 74% higher chance ofdementia. Other studies have demonstrated that an Ad-5 vector that hasbeen used in gene therapy caused inflammation of the brain. Wild typeAd-5 has been reported to cause obesity and we have noted that an Ad-5vector that is used for gene transfer retains its ability to stimulateSREBP-1 and FAS synthesis in hepatocytes. Accordingly, these datasupport the concept that adipogenic adenoviruses may increase lipogenicenzymes in the brain and nerves, produce excess fatty acid andtriglycerides, and result in dysfunction.

An aspect of the invention is that testing for adipogenic adenovirusesmay be used to predict adrenal dysfunction. Also, a vaccine againstadenoviruses may be used to prevent adrenal dysfunction.

Obesity is known to significantly affect adrenal gland function. Thereis an increased secretion of aldosterone, but without renin, which maybe associated with hypertension. Studies have demonstrated that adrenalfunction was not normal in obese women, but differed with abdominal fatdistribution. Adrenal hormones use cholesterol as a substrate forproduction, and with the changes of adipogenic adenoviruses in bothintracellular fatty acids and cholesterol metabolism, the use of a testto detect adipogenic adenoviruses may be a predictor of adrenaldysfunction.

Exemplary screening immunoanalytical techniques include withoutlimitation, standard virus neutralization assay techniques or enzymeimmunoassay techniques well known in the art. Techniques for raising andpurifying antibodies against these viruses or fragments thereof (e.g.,fiber protein or fragments thereof), or proteins (or fragments thereof)from these viruses for use in these immunoassay techniques may beprepared by conventional techniques are well known in the art. In aspecific embodiment of the invention, antibodies will immunoprecipitateadenovirus virus or adenovirus proteins from solution as well as reactwith these proteins on Western or immunoblots or polyacrylamide gels. Inanother specific embodiment, antibodies will detect the presence ofadenovirus or adenovirus proteins in frozen tissue sections, usingimmunocytochemical techniques. Specific embodiments relating to methodsfor detecting adenovirus or adenovirus proteins include enzyme linkedimmunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometricassays (IRMA) and immunoenzymatic assays (IEMA), including sandwichassays using monoclonal and/or polyclonal antibodies.

Similarly, the nucleic acid probe hybridization assay techniques used inthese methods of the invention will be standard techniques (optionallyafter amplification of DNA or RNA extracted from a sample of blood,other body fluid, feces, tissue or organ) using nucleic acid probes (andprimers if amplification is employed) made available by theobesity-causing viruses identified and made available by the presentinvention. The sequences of nucleic acids characteristic of theseviruses can be determined by standard techniques once the viruses areconventionally isolated, and probes and primers that are specific forthe viruses and that provide the basis for nucleic acid probes andprimers that can be used in nucleic acid based assays for the virusesare prepared using conventional techniques on the basis of thesequences.

For example, in order to detect the presence of an adenoviruspredisposing an individual to obesity, a biological sample such as bloodis prepared and analyzed for the presence or absence of adenovirusproteins, such as the Ad-36 fiber coat protein sequences. Results ofthese tests and interpretive information are returned to the health careprovider for communication to the tested individual. Such diagnoses maybe performed by diagnostic laboratories, or alternatively, diagnostickits are manufactured and sold to health care providers or to privateindividuals for self-diagnosis.

Initially, screening involves amplification of the relevant adenovirussequences. In a specific embodiment of the invention, the screeningmethod involves a non-PCR based strategy. Such screening methods includetwo-step label amplification methodologies that are well known in theart. Both PCR and non-PCR based screening strategies can detect targetsequences with a high level of sensitivity.

One embodiment of the invention relates to target amplification. Here,the target nucleic acid sequence is amplified with polymerase. Onespecific method using polymerase-driven amplification is the polymerasechain reaction (PCR). The polymerase chain reaction and otherpolymerase-driven amplification assays can achieve over a million-foldincrease in copy number through the use of polymerase-drivenamplification cycles. Once amplified, the resulting nucleic acid can besequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequencesthe biological sample to be analyzed, such as blood or serum, may betreated, if desired to extract the nucleic acids. The sample nucleicacid may be prepared in various ways to facilitate detection of thetarget sequence, e.g., denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniques wellknown in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the analyte.The region of the probes which is used to bind to the analyte can bemade completely complementary to the targeted region of the adenovirusof interest, and in particular the fiber coat protein. Therefore, highstringency conditions are desirable in order to prevent false positives.However, conditions of high stringency are used only if the probes arecomplementary to regions of the adenovirus. The stringency ofhybridization is determined by a number of factors during hybridizationand during the washing procedure, including temperature, ionic strength,base composition, probe length, and concentration of formamide.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, however, the probe may beunlabeled, but may be detectable by specific binding with a ligand whichis labeled, either directly or indirectly. Suitable labels, and methodfor labeling probes and ligands are well known in the art, and include,for example, radioactive labels which may be incorporated by knownmethods (e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes) enzymes,antibodies, gold nanoparticles and the like. Variations of this basicscheme are known in the art, and include those variations thatfacilitate separation of the hybrids to be detected from extraneousmaterials and/or that amplify the signal from the labeled moiety.

As noted above, non-PCR based screening assays are also contemplated bythis invention. This procedure hybridizes a nucleic acid probe (oranalog such as a methyl phosphonate backbone replacing the normalphosphodiester) to the low level DNA target. This probe may have anenzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Theenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe conjugate and a substrate is added for enzymedetection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in about a 10³ to about a10⁶ increase in sensitivity.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digioxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecific binding the adenovirus sequence region of interest. In oneexample, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digioexigenin is attached to the nucleicacid probe. Hybridization is detected by an antibody-alkalinephosphatase conjugate which turns over a chemiluminescent substrate. Ina second example, the small ligand is recognized by a secondligand-enzyme conjugate that is capable of specifically complexing tothe first ligand. A well known embodiment of this example is thebiotin-avidin type interactions.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting various species ofadenoviruses. Thus, in one example to detect the presence of ad-36,ad-37 and/or ad-5, for example, in a biological sample, more than oneprobe complementary of the targeted regions of interest in the varioustypes of adenovirus may be employed.

As the skilled will understand, more than one strain of obesity-causingvirus may be tested for simultaneously in an immunological or nucleicacid-based assay method for testing for virus in accordance with theinvention and kits may be assembled to facilitate carrying out themethods for a particular virus or a plurality of them.

The invention has been disclosed broadly and illustrated in reference torepresentative embodiments described above. Those skilled in the artwill recognize that various modifications can be made to the presentinvention without departing from the spirit and scope thereof. Withoutfurther elaboration, it is believed that one skilled in the art, usingthe preceding description, can utilize the present invention to thefullest extent. The following examples are illustrative only, and notlimiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES Specific Example 1

Blood samples from 502 humans in separate populations from three cities:Madison, Wis., Naples, Fla., and New York City, N.Y., were measured forAd-36 infection. The definition of obesity was a BMI greater than about30 kg/m² and the population was divided into obese and non-obesesubjects. The prevalence of Ad-36 antibodies in serum obese subjects wasabout 30% and in non-obese was about 11% (i.e., a 3:1 ratio) (FIG. 13).

When the subjects were divided into antibody positive versus antibodynegative, the antibody positive were observed to be about 9 BMI unitsheavier (i.e., greater than about 50 pounds)(p<0.001) (FIG. 14).

Moreover, serum cholesterol and triglycerides were lower in antibodypositive humans (less than about −35 mg/dl, p<0.001), exactly the samereductions observed in prospectively infected monkeys. There were nocorrelations of antibodies to Ad-2 or Ad-31 with either body compositionor serum cholesterol and triglycerides in humans, implying that theeffects of Ad-36 are specific and are not common to all humanadenoviruses.

A second study was performed in 28 twin pairs discordant for Ad-36antibodies. Normally twins track body weight and BMI closely, but theAd-36 antibody positive twins were fatter than their Ad-36 antibodynegative co-twin (p<0.04) (FIG. 15).

In conclusion, these findings show that Ad-36 causes obesity in animalsand humans.

Specific Example 2

Very strong evidence of an association of Ad-36 with cancer comes fromstudies at the University of Wisconsin. Banked serum from 128 subjectswith breast cancer and 37 subjects with prostate cancer was obtained,and assayed for Ad-36 antibodies using a serum neutralization assay. Itwas observed that 51% of the patients with prostate cancer and 50% ofthe breast cancer patients were positive for Ad-36 antibodies. However,since the samples were anonymous, no data on obesity, type or stage ofcancer, or presence of metastases. From data on Ad-36 antibodies inobese and non-obese individuals it was estimated that about 14% of thegeneral population had Ad-36 antibodies. Thus, there is an almost fourfold increase in the prevalence of Ad-36 antibodies in both breast andprostate cancer patients.

Specific Example 3

Prostate cancer tissue from 18 patients was obtained and assayed forAd-36 DNA by nested polymerase chain reaction assay (PCR) using primersmade to unique DNA sequences in the Ad-36 fiber protein genome. Elevenof the 18, or 61%, had Ad-36 DNA in the cancer tissue. FIG. 16 shows aPCR gel demonstrating the presence of Ad-36 DNA in prostate cancertissues. As noted above, Ad-36 DNA in multiple tissues of experimentalanimals was found, particularly in adipose tissue using nested PCR withprimers made from unique DNA sequences in the Ad-36 fiber proteingenome. FIG. 17 shows a PCR gel from DNA extracted from adipose tissueof monkeys infected with Ad-36. All three of the infected monkeys, butnone of the controls, have viral DNA present.

Specific Example 4

This example shows the use of Ad-36 lab test as a marker to identifyindividuals at risk of developing cancer: People with cancer have a muchhigher prevalence of a positive Ad-36 lab test than do non-cancerpatients. We tested 128 women with breast cancer and 37 men withprostate cancer and compared the results to 502 non-cancer patients. Ofthe total of 165 cancer patients, 83 (50%) had a positive Ad-36 test(50% of the breast cancer patients and 51% of the prostate cancerpatients). Based on the prevalence of obesity in the US population, thedata of the 502 non-cancer subjects showed that 17% of the non-cancersubjects had a positive Ad-36 test. If only those non-cancer subjectsfrom Wisconsin are considered (since all the cancer patients were fromWisconsin, this is a better comparison), 14% of the non-cancer subjectshad a positive Ad-36 test. Thus, almost 4 times as many cancer patientswere positive compared to the non-cancer patients.

Specific Example 5

Adipose tissue from 9 humans at autopsy at the University of WisconsinHospital was obtained and assayed for Ad-36 DNA. Seven of the 9 hadAd-36 DNA in the adipose tissue. These data demonstrate that Ad-36 DNAmay isolated from human and animal adipose tissue and more importantly,that the presence of Ad-36 DNA in tissues is a marker for post Ad-36infection. Since the Ad-36 DNA appeared in multiple tissues of infectedanimals, it seems clear that the initial viremia results in infection ofmost of the tissues of the body. Therefore, as described in the methodsSection as described in Specific Example 6, below, cancer tissues oradipose tissue from cancer may be obtained from patients to test for thepresence of Ad-36 DNA.

Specific Example 6

Samples are assayed for Ad-36 DNA in samples of breast and prostatetissue from cancer patients and non-cancer patients to determine if theprevalence of infection with Ad-36 is greater in cancer patients. Ad-36status may be correlated with the presence of cancer and may becorrelated with stage of cancer, presence of metastases, and prognosisof cancer victims. A vaccine against Ad-36 may prevent Ad-36 inducedcancers.

Experimental Design:

Anonymous samples of tissue and complete medical information areobtained from the NCI resource banks, the NCI Cooperative Human TissueNetwork (CHTN) and the NCI Cooperative Breast Cancer Tissue Resource(CBCTR). The samples are assayed for Ad-36 DNA by nested PCR assay asdescribed below. Alternatively, if DNA samples not are available, theyare may ordered. If DNA has not been extracted from the samples, it maybe as described below.

To evaluate breast cancer, samples of breast cancer tissue or adiposetissue near the breast from 100 women with cancer are compared withbreast tissue or adipose tissue from 50 women undergoing breast surgerywho do not have cancer. As noted above, if breast cancer tissue is notavailable, adipose tissue from near the breast is acceptable. Actualbreast tissue is not critical as Ad-36 DNA appears in all tissues afterinfection, so adipose tissue is suitable.

To evaluate prostate cancer, samples of prostate cancer from 100 men iscompared to prostate tissue from men undergoing surgery for benignprostatic hypertrophy (BPH).

Both NCI tissue banks have information on age, menopausal status offemale patients, height, weight, family history of breast or prostatecancer, type and stage of cancer, presence of metastases, and in somecases, survival time. Because breast cancer associated with obesity ismore prevalent in postmenopausal women, non-cancer subjects are matchedas well as possible for age. It may be expected that prostate cancerpatients from whom we obtain samples will be in the older age group (>50years) because this is the population in which prostate cancer and BPHoccurs. It may be very unlikely that we will have any tissues frompediatric patients as breast and prostate cancers are so rare in them.

Laboratory techniques: The procedures that will be needed for thisprotocol are published in the medical literature are understood by oneof relevant skill in the art. A brief summary of each is given below:

Culture of Ad-36: Viral preparations are grown in tissue culture aspreviously described using A549 human bronchial carcinoma cells. A549cells are obtained from American Type Culture Collection (ATCC,Rockville, Md.). Minimum Essential Media Eagle (MEM) (Cat #M-0643, SigmaChemicals) with non-essential amino acids, Earle's salts and L-glutamineare used for growing A549 cells. Work stocks are grown in A549 cellsusing MEM with 10% fetal bovine serum (FBS) and 2.9% NaHCO₃ (v/v) at pH7.4.

Plaque forming units assay: Titers of Ad-36 virus are determined usingA549 cells by this assay. Starting with 100 μl of virus suspension and900 μl of media, serial 10 fold dilutions are made. A549 cells are grownto confluence in 6 well plates and 3 wells are used for each dilution.Three wells are used as the blank control and are not infected with thevirus suspension. Media is removed from each cell and 100 ul of theserially diluted virus suspension are pipetted out in the wells. Theplates are incubated at 37° C., shaking gently every 15 min. After 1 hof incubation, the viral suspension from the wells is removed anddiscarded. The wells are overlaid with about 3 ml of 1% agar in mediaper well, with 1× antibiotic-antimycotic solution. The plates areinverted and incubated at 37° C. for 8 days until plaques appear. After8 days, wells are stained overnight with about 1 ml of crystal violetper well. The next day, the number of plaques formed is counted afterremoving the agar. The number of plaques formed×dilution of viralsuspension used gives PFU/100 uL of inoculum used. This is multiplied by10 to express PFU/mL.

Tissue culture infectivity dose (TCID50)(66-72): The titer of the workstocks that cause a cytopathic effect (CPE) in 50% of the wellscontaining A549 cells are calculated and expressed as tissue cultureinfectivity dose (TCID-50) units per ml. TCID-50 of the work stocks aredetermined using serial ten fold dilutions of the virus work stock.TCID50 is calculated by serially diluting the virus stock solution andinoculating cells with the dilutions to find out the reciprocal of thehighest dilution of virus which causes cytopathic effect (CPE) in 50% ofthe cells inoculated. Titers are calculated after the cells inoculatedwith the virus dilutions are incubated at 37° C. for 8 days.

Nested PCR assay: The nested PCR detects Ad-36 DNA in biologicalsamples. As previously described, four primers were designed to uniqueregions of the Ad-36 fiber protein gene for use in a nested PCR assayfor detection of viral DNA. Sequences of primers:

outer forward primer (5′-GTCTGGAAAACTGAGTGTGGATA), (SEQ ID No:1) outerreverse primer (5 = -ATCCAAAATCAAATGTAATAGAGT), (SEQ ID No:2) innerforward primer (5 = -TTAACTGGAAAAGGAATAGGTA), (SEQ ID No.3) innerreverse primer (5 = -GGTGTTGTTGGTTGGCTTAGGATA). (SEQ ID No.4)

DNA is isolated from human breast and prostate tissues using a QIAampTissue Kit (Qiagen, Valencia, Calif., USA; Cat #29304). Negative PCRcontrols are water and DNA from A549 cells. Positive PCR control is DNAfrom Ad-36 infected A-549 cells. DNA is denatured for 2 min at 95° C.and subjected to 35 cycles of PCR (94° C. for 1 min, 55° C. for 1 min,72° C. for 2 min) followed by incubation at 72° C. for 5 min. PCRproducts are visualized on a 1% agarose gel with a size marker.

This assay was developed at the University of Wisconsin and at that timethe conditions were optimized for temperature, magnesium concentration,and number of cycles in samples consisting of animal and human tissues,and of 3T3-L1 cells. Samples of the PCR products are extracted from thegels and are sent to the University of Wisconsin Biotech Center forsequencing to insure that the amplified DNA sequences were from thetargeted regions. The Biotech Center confirmed their accuracy. Thesequality control procedures are repeated to conform to CLIAspecifications and good laboratory practice. DNA sequencing of twopositive samples each of breast cancer and of prostate cancer and theA549 positive control DNA samples are performed to insure accuracy ofthe amplifications.

Statistical analyses and Power calculations: Statistical assistance isavailable from the Department of Statistical Sciences and OperationsResearch of the Virginia Commonwealth University.

Power calculations: Power analysis was performed using preliminary dataon the prevalence of Ad-36 antibodies in the general population versusin cancer patients in Madison, Wis. It was shown that 50% and 51% ofbreast cancer and prostate cancer patients, respectively, at theUniversity of Wisconsin Hospital had antibodies to Ad-36. In a sample ofvolunteers from the general population and the Obesity Treatment Programat the University of Wisconsin, 247 subjects were evaluated, of whom 183were obese and 64 were non-obese. The prevalence of Ad-36 antibodies was20% and 11%, respectively. CDC figures show that approximately 31% ofadult Americans are obese and 69% are non-obese. Using these numbers, itwas estimated that the prevalence of Ad-36 antibodies in the generalpopulation of Madison, Wis., was 13.8%. Thus, the prevalence of Ad-36 incancer patients is almost fourfold higher. Using these figures, thepower calculations revealed that 32 subjects per group is necessary toachieve a power of 80% at the 0.05 level to determine a difference inprevalence of Ad-36 in cancer vs non-cancer patients. About 50% of bothbreast and prostate cancer patients may be expected to be Ad-36 antibodypositive based on the preliminary data. However, it may be possible thatthe Ad-36 prevalence may be lower in the banked samples from the NCItissue banks (or Asterand tissue bank), and this would affect the powersignificantly. Therefore, 100 cancer subjects and 50 non-cancer subjectsmay be certain to have sufficient power.

Statistical analyses: Chi-square analysis will be used to determine ifthe prevalence of Ad-36 DNA in cancer subjects is greater than innon-cancer subjects. The number of subjects is relatively small formultiple regression analyses and we do not expect to see significantcorrelations unless the effect is powerful.

Specific Example 7 Example 8

The cDNA sequence of the Ad-36 genome was screened against all knowncDNA sequences and two 25-base sequences and one 28-base sequence werefound, all lying in the fiber-encoding sequence that were unique toAd-36. These three sequences are as follows:

SEQ ID NO: 5: 5′-AGTTGAAACAGCAAGAGACTCAAAG SEQ ID NO: 65′-GGTACTGGATCAAGTGCACATGGAG SEQ ID NO: 75′-TTGAAACAGCAAGAGACTCAAAGCTAAC

Sequence 3 above was employed a probe for Ad-36 in a conventional nucleiacid probe hybridization assay of DNA isolated from four chickens, twoof which had been infected with the virus and became obese and two ofwhich had not been infected and were not obese. DNA hybridizing to theprobe was observed with only the DNA from the two infected chickens. Theassay involved direct detection and was by capillary electrophoresisusing laser-induced fluorescence for detection. More particularly, areplaceable polyacrylamide matrix was employed in the electrophoreticseparation and detection employed a dual system with 5′-labeling of theoligo and thiazole orange intercalator in the buffer system.

The skilled will understand that probes, and primers when amplificationis also used, of between about 15 and 30 bases in length areadvantageously employed to provide suitable specificity and sensitivity.Amplification methods using PCR and variations thereof maybe employed,as well known in the art.

The examples given above are merely illustrative and are not meant to bean exhaustive list of all possible embodiments, applications ormodifications of the invention. Thus, various modifications andvariations of the described methods and systems of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the appended claims.

The disclosures of all references and publications cited above areexpressly incorporated by reference in their entireties to the sameextent as if each were incorporated by reference individually.

1. A method for determining if a subject is predisposed to developing anobesity-related disease due to an adipogenic adenovirus, said methodcomprising the steps of: obtaining a sample from the subject; assayingthe sample to determine whether the subject is infected with anadipogenic adenovirus; and determining that a subject infected with anadipogenic adenovirus is predisposed to developing an obesity relateddisease due to an adipogenic adenovirus relative to a subject notinfected with an adipogenic adenovirus.
 2. The method of claim 1,wherein said step of assaying the sample comprises the steps of:screening the sample for the presence of antibodies specific to theadipogenic adenovirus in the sample; and determining the presence ofantibodies specific to the adipogenic adenovirus in the sample.
 3. Themethod of claim 1, wherein the adipogenic adenovirus is one or moreadipogenic viruses selected from the group consisting of adenovirus type5, adenovirus type 36, and adenovirus type
 37. 4. The method of claim 1,wherein the obesity-related disease is a disease selected from the groupconsisting of cancer, prostrate cancer, breast cancer, pancreaticdysfunction, pancreatic disease, diabetes, insulin resistance, metabolicdysregulation, pulmonary disease, pulmonary dysfunction, brain andnervous system dysfunction, liver disease, muscle dysfunction, cardiacdisease, cardiac dysfunction, gall bladder diseases, hypertension,hyperlipoproteinemia, toxemia during pregnancy, decreased fertility,gout, arthritis, and adrenal dysfunction.
 5. The method of claim 4,wherein the pulmonary disease is selected from the group consisting ofsleep apnea, asthma, and emphysema.
 6. The method of claim 4, whereinthe cardiac disease is selected from the group consisting ofatherosclerotic disease and congestive heart failure.
 7. The method ofclaim 4, wherein the liver disease is selected from the group consistingof steatohepatitis, non-alcoholic steatohepatitis, and cirrhosis.
 8. Themethod of claim 4, wherein the gall bladder disease is selected from thegroup consisting of gallstones and cholecystitis.
 9. The method of claim4, wherein the brain and nervous system dysfunction is selected from thegroup consisting of Alzheimer's disease and stroke.
 10. The method ofclaim 4, wherein the muscle dysfunction is selected from the groupconsisting of intramuscular lipid accumulation and skeletal muscle lipidaccumulation.
 11. The method of claim 1, wherein the subject is a human.12. The method of claim 1, wherein the subject is an animal.
 13. Themethod of claim 2, wherein the antibodies in said determining step arespecific to one or more peptide encoded by the nucleic acid sequencesselected from the group consisting of SEQ ID NO.:5, SEQ ID NO.:6, andSEQ ID NO.:7.
 14. The method of claim 2, wherein said screening step isperformed by using an immunoanalytical technique.
 15. The method ofclaim 14, wherein said immunoanalytical technique is one or moretechniques selected from the group consisting of serum neutralizationassay and enzyme immunoassay.
 16. The method of claim 15, wherein saidenzyme immunoassay is selected from the group consisting of ELISA, RIA,IRMA, IEMA, and immunocytochemical assay.
 17. The method of claim 1,wherein the sample is selected from the group consisting of a biologicalsample, body fluid, a tissue sample, an organ sample, feces, blood,saliva, and any combination thereof.
 18. The method of claim 2, whereinthe presence of antibodies specific to the adipogenic adenovirus in thesample is indicative of a subject being predisposed to developing anobesity-related disease due to adipogenic adenovirus.
 19. The method ofclaim 1, wherein the subject infected with the adipogenic adenovirus hasincreased production of lipogenic enzymes in comparison to a subject notinfected with the adipogenic adenovirus.
 20. The method of claim 19,wherein lipogenic enzymes are one or more enzymes selected from thegroup consisting of FAS, glycerol-3-phosphodehydrogenase, lipoproteinlipase, SCD1, CPT 1, and L-type pyruvate kinase.
 21. The method of claim1, wherein said step of assaying the sample comprises the steps of:screening the sample for the presence of nucleic acid specific to theadipogenic adenovirus in the sample; and determining the presence ofnucleic acid specific to the adipogenic adenovirus in the sample. 22.The method of claim 21, wherein said step of screening is performed byusing a nucleic acid hybridization technique.
 23. The method of claim22, wherein said nucleic acid hybridization technique is selected fromthe group consisting of direct DNA sequencing, RNA sequencing, RNaseprotection assays, PCR, RT-PCR, real time PCR, SI nuclease protectionassay, nucleic acid affinity purification, and nucleic acidhybridization.
 24. The method of claim 21, wherein the nucleic acid isDNA selected from the group consisting of cDNA, ssDNA, dsDNA, andgenomic DNA.
 25. The method of claim 21, wherein the nucleic acid is RNAselected from the group consisting of mRNA, dsRNA, and ssRNA.
 26. Themethod of claim 21, wherein the presence of nucleic acid specific to theadipogenic adenovirus in the sample is indicative of a subject beingpredisposed to developing an obesity-related disease due to adipogenicadenovirus.